HAVCR2 Mouse

What is the molecular structure of mouse HAVCR2/TIM-3?

Mouse HAVCR2/TIM-3 is a member of the immunoglobulin superfamily and TIM family of proteins. The protein contains an extracellular domain spanning from Leu22 to Arg191. The full recombinant protein has a predicted molecular weight of 19.9 kDa, but due to significant glycosylation, it typically migrates to 40-68 kDa when analyzed by Bis-Tris PAGE . The protein structure includes immunoglobulin and mucin domains that are critical for its binding interactions and downstream signaling functions .

How does mouse HAVCR2/TIM-3 compare structurally to human HAVCR2/TIM-3?

While both mouse and human HAVCR2/TIM-3 belong to the same protein family and share functional similarities, there are important structural differences to consider when designing cross-species experiments. Both contain immunoglobulin and mucin domains, but amino acid sequence variations exist that may affect binding affinities to ligands. This is particularly important when testing therapeutic antibodies or ligand interactions that may not translate directly between species .

What are the common synonyms and accession numbers for mouse HAVCR2?

Mouse HAVCR2 is commonly referenced using several alternative identifiers:

• TIM-3 or Tim-3

• Timd3

• CD366

• Q8VIM0-1 (UniProt accession)

• MGI:2159682 (Mouse Genome Informatics ID)

These alternative designations are important to know when conducting literature searches or database queries to ensure comprehensive coverage of relevant research.

What is the expression pattern of HAVCR2/TIM-3 in mouse immune cells?

Mouse HAVCR2/TIM-3 is predominantly expressed on several immune cell populations:

• CD4+ Th1 cells but not Th2 cells

• Exhausted CD8+ T cells, particularly in chronic infection and cancer contexts

• Natural killer (NK) cells

• Dendritic cells

• Regulatory T cells (Treg)

• Myeloid-derived suppressor cells

The expression is typically induced following T cell activation and is considered a marker of T cell exhaustion when co-expressed with other checkpoint molecules like PD-1 .

How is HAVCR2/TIM-3 expression regulated in mouse models?

HAVCR2/TIM-3 expression is dynamically regulated in response to various stimuli. In T cells, its expression increases following activation and during chronic antigen exposure, correlating with an exhausted phenotype. The regulation involves transcriptional mechanisms that can be influenced by cytokine signaling pathways, particularly those associated with Th1 differentiation. In dendritic cells, expression can be modulated by exposure to danger-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) .

What is known about tissue-specific expression of mouse HAVCR2?

According to the International Mouse Phenotyping Consortium (IMPC) data, expression of HAVCR2 has been examined in 73 adult tissues, though specific lacZ expression data appears to be limited . The protein is predominantly expressed in immune tissues and cells rather than showing broad tissue distribution. Its expression in non-immune tissues under pathological conditions remains an active area of investigation.

What are the primary ligands of mouse HAVCR2/TIM-3 and their binding properties?

Mouse HAVCR2/TIM-3 interacts with several ligands that mediate its diverse functions:

• Galectin-9: A primary binding partner that induces immunosuppressive functions. The binding enhances immune tolerance and inhibits anti-tumor immunity. The ED50 for binding to human Galectin-9 in functional ELISA is less than 20 μg/ml .

• Phosphatidylserine (PtSer): HAVCR2/TIM-3 can recognize phosphatidylserine on apoptotic cells in a calcium-dependent manner, facilitating their phagocytosis, particularly by dendritic cells .

• CEACAM-1: Heterodimerization with CEACAM-1 appears critical for the inhibitory function of TIM-3. Co-blockade of TIM-3 and CEACAM-1 has shown enhanced antitumor responses in mouse models .

• HMGB1: In tumor-infiltrating dendritic cells, TIM-3 can interact with HMGB1, interfering with nucleic acid sensing and trafficking to endosomes .

How does HAVCR2/TIM-3 signaling affect T cell function in mouse models?

HAVCR2/TIM-3 signaling in mouse T cells has complex effects:

• Inhibitory Functions: Generally accepted to have an inhibitory role, TIM-3 attenuates TCR-induced signaling, specifically by blocking NF-κB and NFAT promoter activities, resulting in reduced IL-2 secretion. This function may involve its association with LCK, which impairs phosphorylation of TCR subunits .

• T Cell Exhaustion: When co-expressed with PD-1 on tumor-infiltrating T cells, TIM-3 contributes to a severe exhausted phenotype, characterized by reduced proliferation and cytokine production .

• Apoptosis Induction: Binding to Galectin-9 can induce apoptosis of antigen-specific T cells, potentially involving TIM-3 phosphorylation and disruption of its association with BAG6 .

• Context-Dependent Activation: In contrast to its inhibitory functions, TIM-3 has also been shown to activate TCR-induced signaling in some contexts, potentially involving ZAP70, LCP2, LCK, and FYN .

What role does HAVCR2/TIM-3 play in dendritic cell function in mice?

In mouse dendritic cells (DCs), HAVCR2/TIM-3 has several important functions:

• Phagocytosis of Apoptotic Cells: TIM-3 mediates the engulfment of apoptotic cells by DCs through recognition of phosphatidylserine .

• Antigen Cross-Presentation: TIM-3 facilitates the cross-presentation of antigens from apoptotic cells .

• Immunoregulation: TIM-3 expressed on DCs can positively regulate innate immune responses and, in synergy with Toll-like receptors, promote TNF-α secretion .

• Suppression of Nucleic Acid Sensing: In tumor-infiltrating DCs, TIM-3 can suppress nucleic acid-mediated innate immune responses by interacting with HMGB1 and interfering with nucleic acid sensing and trafficking to endosomes .

What are the optimal conditions for reconstituting and storing recombinant mouse HAVCR2/TIM-3 protein?

For optimal handling of recombinant mouse HAVCR2/TIM-3 protein:

• Reconstitution:

  • Centrifuge the tube before opening

  • Reconstitute lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Adding 5-50% glycerol (final concentration) is recommended for long-term storage

• Storage Conditions:

  • Store at -20°C to -80°C for 12 months as supplied from date of receipt

  • Storage at -80°C for 3 months after reconstitution

  • Aliquot the protein into smaller quantities for optimal storage

  • Minimize freeze-thaw cycles as repeated freezing and thawing is not recommended

  • Working aliquots can be stored at 4°C for up to one week

What experimental approaches can be used to study HAVCR2/TIM-3 function in mouse models?

Several methodological approaches are effective for studying mouse HAVCR2/TIM-3:

• Genetic Models:

  • HAVCR2 knockout mice (available through resources like IMPC)

  • Conditional knockout models for tissue-specific deletion

  • Transgenic overexpression models

• Protein Interaction Studies:

  • Functional ELISA to assess binding to ligands like Galectin-9

  • Co-immunoprecipitation to identify binding partners

  • Surface plasmon resonance for kinetic binding studies

• Functional Assays:

  • T cell exhaustion assessments (proliferation, cytokine production)

  • Apoptosis assays following TIM-3 ligation

  • Phagocytosis assays for dendritic cells

  • Cytotoxicity assays for NK cells

• In vivo Models:

  • Chronic infection models to study T cell exhaustion

  • Cancer models to assess tumor-infiltrating lymphocyte function

  • Autoimmune disease models to evaluate inflammatory responses

What are the common challenges in studying mouse HAVCR2/TIM-3 and how can they be addressed?

Researchers often encounter several challenges when studying mouse HAVCR2/TIM-3:

• Protein Glycosylation Variability:

  • Challenge: The extensive glycosylation of TIM-3 (causing migration at 40-68 kDa despite a predicted MW of 19.9 kDa) can affect antibody recognition and functional properties.

  • Solution: Use deglycosylation enzymes for certain applications and validate antibodies with appropriate controls .

• Context-Dependent Functions:

  • Challenge: TIM-3 can have both inhibitory and stimulatory functions depending on cellular context.

  • Solution: Clearly define the cellular system and provide comprehensive contextual information when reporting results .

• Ligand Specificity:

  • Challenge: Some ligand interactions (like with Galectin-9) have been challenged in the literature.

  • Solution: Use multiple methodological approaches to confirm ligand interactions and consider complementary functional readouts .

• Species Differences:

  • Challenge: Mouse and human TIM-3 have structural and functional differences.

  • Solution: Validate findings across species when possible and be cautious about direct translational implications .

How does HAVCR2/TIM-3 contribute to immune responses in mouse cancer models?

In mouse cancer models, HAVCR2/TIM-3 plays several important roles:

• T Cell Exhaustion: TIM-3 is expressed by exhausted T cells in cancer settings. Tumor-infiltrating T cells that co-express PD-1 and TIM-3 exhibit the most severe exhausted phenotype, characterized by diminished anti-tumor activity .

• Dendritic Cell Dysfunction: TIM-3 expression on tumor-infiltrating dendritic cells suppresses innate immunity by reducing the immunogenicity of nucleic acids released by dying tumor cells, thereby contributing to immune evasion .

• Regulatory T Cell Function: TIM-3 expressed on Treg cells can inhibit effector T cell responses, including Th17 responses, further dampening anti-tumor immunity .

• Therapeutic Target: Blockade of TIM-3, particularly in combination with other checkpoint inhibitors, has shown promise in enhancing anti-tumor responses in various mouse cancer models .

What is known about HAVCR2/TIM-3 in mouse models of chronic infection?

HAVCR2/TIM-3 has significant implications in chronic infection models:

• T Cell Exhaustion Marker: Similar to cancer contexts, TIM-3 is upregulated on exhausted T cells during chronic viral infections, contributing to impaired clearance of persistent pathogens .

• Antimicrobial Responses: TIM-3 expressed on Th1 cells interacts with Galectin-9 on Mycobacterium tuberculosis-infected macrophages to stimulate antibactericidal activity, including IL-1β secretion, and to restrict intracellular bacterial growth .

• Acute Infection Responses: TIM-3 has been reported to enhance CD8+ T cell responses to acute infections such as Listeria monocytogenes, highlighting its context-dependent functionality .

• NK Cell Regulation: TIM-3 expressed on NK cells can enhance IFN-γ production in response to certain pathogens but can also suppress NK cell-mediated cytotoxicity in other contexts, demonstrating its complex role in innate immunity .

What phenotypes are observed in HAVCR2/TIM-3 knockout mouse models?

According to the International Mouse Phenotyping Consortium (IMPC), HAVCR2 knockout mice exhibit specific phenotypes:

• Significant Phenotypes: Two significant phenotypes have been identified in HAVCR2 knockout mice, though detailed descriptions are not provided in the available search results .

• Associated Diseases: Two diseases are associated with HAVCR2 mutations, based on comparisons of mouse phenotypes with human disease phenotypes .

• Physiological Systems Impact: The knockout appears to significantly impact certain physiological systems, though specific details would require further investigation .

• Autoimmunity Predisposition: Based on the known functions of TIM-3 in regulating T cell responses, knockout mice may exhibit enhanced susceptibility to autoimmune conditions due to dysregulated Th1 responses .

How might the heterodimeric interactions of HAVCR2/TIM-3 be exploited for therapeutic development?

The interaction between HAVCR2/TIM-3 and other molecules, particularly CEACAM-1, presents interesting therapeutic opportunities:

• Co-targeting Strategies: Research indicates that heterodimerization of TIM-3 with CEACAM-1 is critical for the inhibitory function of TIM-3. Co-blockade of TIM-3 and CEACAM-1 has enhanced antitumor responses in a mouse model of colorectal cancer .

• Structure-Based Drug Design: Understanding the structural basis of these heterodimeric interactions could inform the development of small molecules or biologics that specifically disrupt these interactions while preserving beneficial TIM-3 functions.

• Combination Therapies: Exploring combinations of TIM-3 blockade with other checkpoint inhibitors (beyond PD-1) based on heterodimeric interactions could identify synergistic therapeutic approaches for cancer immunotherapy.

• Cell Type-Specific Targeting: Developing strategies to target TIM-3 heterodimeric complexes on specific cell populations (e.g., tumor-infiltrating dendritic cells vs. T cells) might improve therapeutic specificity and reduce off-target effects.

What are the implications of HAVCR2/TIM-3 phosphatidylserine binding for efferocytosis research?

The capacity of HAVCR2/TIM-3 to bind phosphatidylserine (PtSer) on apoptotic cells has significant implications for efferocytosis research:

• Mechanistic Studies: Investigating the calcium-dependent binding mechanism of TIM-3 to PtSer could illuminate general principles of phospholipid recognition by immune receptors.

• Cell Type-Specific Effects: While TIM-3 on dendritic cells mediates engulfment of apoptotic cells, TIM-3 on T cells promotes conjugation but not engulfment. Understanding this differential response could reveal important cell-specific signaling mechanisms .

• Cross-Presentation Pathways: Exploring how TIM-3-mediated uptake of apoptotic cells influences antigen processing and cross-presentation pathways in dendritic cells could inform vaccine design and cancer immunotherapy approaches.

• Resolution of Inflammation: Investigating the role of TIM-3 in efferocytosis during the resolution phase of inflammatory responses could identify new approaches for treating inflammatory diseases.

How might single-cell analysis technologies advance our understanding of HAVCR2/TIM-3 biology?

Single-cell technologies offer powerful approaches to address current knowledge gaps in HAVCR2/TIM-3 biology:

• Heterogeneity in Expression Patterns: Single-cell RNA sequencing could reveal previously unrecognized heterogeneity in TIM-3 expression across immune cell populations and within conventionally defined subsets.

• Temporal Dynamics: Single-cell trajectory analysis could elucidate the temporal dynamics of TIM-3 expression during T cell activation, exhaustion, and memory formation.

• Spatial Context: Spatial transcriptomics approaches could map TIM-3 expression in relation to other immune cells within tissues, particularly in tumor microenvironments and sites of chronic infection.

• Protein-Protein Interactions: Single-cell proteomics and proximity labeling approaches could identify cell type-specific protein interaction networks for TIM-3, potentially revealing new binding partners and signaling mechanisms.

What are the most critical experimental controls when studying mouse HAVCR2/TIM-3?

When designing experiments involving mouse HAVCR2/TIM-3, researchers should consider the following critical controls:

• Specificity Controls: Include isotype controls for antibodies and use HAVCR2 knockout cells or tissues to confirm signal specificity.

• Glycosylation Considerations: Given the extensive glycosylation of TIM-3, researchers should consider how glycosylation status might affect experimental outcomes, particularly in binding studies.

• Context Documentation: Clearly document the cellular context and activation state, as TIM-3 functions can vary significantly depending on the cell type and environmental conditions.

• Ligand Validation: When studying ligand interactions, validate binding using multiple methodologies given controversies surrounding some reported TIM-3 ligands.

• Cross-Species Validation: For translational studies, validate key findings across mouse and human systems to account for species-specific differences in TIM-3 biology.

What emerging technologies might advance mouse HAVCR2/TIM-3 research?

Several emerging technologies hold promise for advancing our understanding of mouse HAVCR2/TIM-3:

• CRISPR-Based Screening: Genome-wide CRISPR screens in primary mouse immune cells could identify novel regulators of TIM-3 expression and function.

• Intravital Imaging: Advanced intravital microscopy techniques could track TIM-3-expressing cells in vivo during immune responses, providing insights into dynamic cellular interactions.

• Protein Engineering: Engineered variants of TIM-3 with altered binding properties could help dissect the relative contributions of different ligand interactions to TIM-3 function.

• Organoid Systems: Immune-competent organoid systems could provide more physiologically relevant platforms for studying TIM-3 in tissue-specific contexts.

• Systems Biology Approaches: Integrative computational analyses combining transcriptomic, proteomic, and functional data could reveal emergent properties of TIM-3 signaling networks not apparent from reductionist approaches.

The continued investigation of HAVCR2/TIM-3 in mouse models remains essential for understanding fundamental immunological processes and developing novel therapeutic approaches for cancer, chronic infections, and autoimmune diseases.